The promoter of the constitutively expressed human dihydrofolate reductase (DHFR) gene has been characterized by deletional analyses. Various deletional mutants of the promoter sequence were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and introduced into Hela cells. The relative activity of the CAT constructs was determined by a newly developed quantitative assay system. Two separate positive regulatory elements, from-612 to -360 and from-111 to -72, relative to the major cap site, were identified in the 5' flanking region. A nuclear runoff assay revealed that the transcriptional rate of exon 1 is about 10 fold higher than that of exon 2, indicating that the transcriptional elongation is blocked within intron 1 sequence. This evidence and the characteristic chromatin structure of this region previously defined suggest the possibility that the expression of the DHFR gene is regulated at the level of mRNA elongation during the cell cycle.